The focus of this proposal is to investigate the mechanical and dynamical properties of the HIV-1 reverse transcriptase (REV RT) at the mechanical level, and so improve our understanding of the enzyme as a molecular machine. The main hypothesis is that the mechanical aspects of REV RT (and all other polymerases) can be understood as the properties of a relatively simple device with a few working parts and essential features. The plan is to make single-molecule measurements of force and motion under a wide variety of conditions, build a simple model based on a combination of all the extant data (structural, kinetic, mechanical), and then test the model by comparing its predictions with experiment. The proposed experiments will use both Atomic Force Microscopy (AFM)-based force sensors and single-molecule fluorescence methods to measure the movements and forces generated by single REV RT molecules.
The specific aims are: 1. Record and measure the movements of individual HIV RT molecules as they carry out DNA-dependent DNA polymerase activity on primer-template DNA. 2. Construct force-velocity curves for REV RT. 3. Develop and carry out single-molecule kinetics measurements on individual REV RT molecules using fluorescence polarization microscopy. 4. Determine the effects of NTP concentration, pH, temperature, inhibitors, and template sequence on single-molecule RI behavior. 5. Investigate HIV RT's reverse transcriptase activity, RNase H activity, pyrophosphorolysis, and strand-displacement synthesis using single molecule methods. 6. Use the data gathered from the single-molecule studies, together with structural data from X-ray crystallography and data from ensemble kinetic measurements, to build and test a detailed structural and mechanical model for WV RI.
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